Being “caught in the act” isn’t usually a good thing, but catching DNA methyltransferases in the act is a very good thing for epigeneticists. A team at the University of Southampton (U.K.) now reports that they have developed a reproducible, real-time fluorescent assay that will help scientists understand how these enzymes regulate themselves, interact with proteins, or even screen for methyltransferase inhibitors that could be used as anti-cancer drugs. In fact, it is the first real-time assay to catch methyltransferases red-handed doing their thing to CpGs.
The group modified the break light oligonucleotide assay that they originally developed to monitor adenine methyltransferase activity. Basically, the break light oligo is in a hairpin-loop structure with a fluorophore and quencher close to each other at either end of the molecule. In that form it won’t light up, but cutting the oligo separates the fluorophore and quencher, increasing fluorescence.
In the new assay, the oligo sequence contains four CpGs—three of which are already methylated. When the fourth one gets a methyl group, it becomes a perfect substrate for GlaI, which chops it, igniting the fluorophore. The researchers demonstrated the usefulness of this assay, using it to detect DNMT1 activity and to measure the kinetics of the bacterial cytosine-C5 methyltransferase M.SssI.
To see all the enlightening details, check out the article at Nucleic Acids Research, February 5, 2010.